scholarly journals Complex regulation of AprA metalloprotease in Pseudomonas fluorescens M114: evidence for the involvement of iron, the ECF sigma factor, PbrA and pseudobactin M114 siderophore

Microbiology ◽  
2006 ◽  
Vol 152 (1) ◽  
pp. 29-42 ◽  
Author(s):  
Bláithín Maunsell ◽  
Claire Adams ◽  
Fergal O'Gara

In the soil bacterium Pseudomonas fluorescens M114, extracellular proteolytic activity and fluorescent siderophore (pseudobactin M114) production were previously shown to be co-ordinately negatively regulated in response to environmental iron levels. An iron-starvation extracytoplasmic function sigma factor, PbrA, required for the transcription of siderophore biosynthetic genes, was also implicated in M114 protease regulation. The current study centred on the characterization and genetic regulation of the gene(s) responsible for protease production in M114. A serralysin-type metalloprotease gene, aprA, was identified and found to encode the major, if not only, extracellular protease produced by this strain. The expression of aprA and its protein product were found to be subject to complex regulation. Transcription analysis confirmed that PbrA was required for full aprA transcription under low iron conditions, while the ferric uptake regulator, Fur, was implicated in aprA repression under high iron conditions. Interestingly, the iron regulation of AprA was dependent on culture conditions, with PbrA-independent AprA-mediated proteolytic activity observed on skim milk agar supplemented with yeast extract, when supplied with iron or purified pseudobactin M114. These effects were not observed on skim milk agar without yeast extract. PbrA-independent aprA expression was also observed from a truncated transcriptional fusion when grown in sucrose asparagine tryptone broth supplied with iron or purified pseudobactin M114. Thus, experimental evidence suggested that iron mediated its effects via transcriptional activation by PbrA under low iron conditions, while an as-yet-unidentified sigma factor(s) may be required for the PbrA-independent aprA expression and AprA proteolytic activity induced by siderophore and iron.

2016 ◽  
Vol 78 (6-7) ◽  
Author(s):  
Zaidah Zainal Ariffin ◽  
Mohd Sidek Ahmad ◽  
Rosalia Pepi ◽  
Zainon Mohd Noor

Microbial enzymes have continued to assist diverse reactions as biocatalysts. Soil derived microbes offer a prospective resource for such enzymes. Screening and isolation of proteolytic fungi were carried out from soil sample of a Malaysian virgin forest. Four isolates showed clear zone of protein hydrolysis on skim milk agar representing proteolytic activity. Aspergillus flavus UOA/HCPF 5774 exhibited the highest proteolytic activity with a clear zone diameter of 21 mm followed by Aspergillus niger and Trichoderma harzianum both with a clear zone of 16 mm, and Penicillium simplicissimum strain LP42 with a13 mm clear zone. Crude protease activity of 0.230 – 0.277 Units / ml for each fungus was seen after 24 hours incubation. A decline of protease production was observed after 48 hours incubation except for Aspergillus flavus UOA/HCPF 5774 which showed a drop only after 72 hours incubation. The protease producing fungi were partially identified based on their morphological characteristics, macroscopic and microscopic identification. The identification was confirmed by 18S rRNA Sequence Analysis. The four fungi protease producers were Aspergillus niger, Penicillium simplicissimum strain LP42, Aspergillus flavus UOA/HCPF 5774 and Trichoderma harzianum.


1982 ◽  
Vol 45 (13) ◽  
pp. 1242-1244 ◽  
Author(s):  
A. W. KOTULA ◽  
S. G. CAMPANO ◽  
D. M. KINSMAN

This study evaluated the proteolytic and lipolytic activity of several strains of Thamnidium elegans, Mucor mucedo and Chaetostylum fresenii on selected test proteins and lipids. At 18°C, the zone of hydrolysis to colony size ratio on skim milk agar, representing proteolytic activity after 4 d, was 0.92, 0.80 and 0.67 for M. mucedo, C. fresenii and T. elegans, respectively. A similar trend was noted after 4 d of incubation at 24°C. There was positive lipolytic activity on Tween 80 at 18 and 24°C for the same three molds. The proteolytic and lipolytic activity decreased with decreasing temperatures so that at 4°C, the temperature of most probable use if applied to meat, the effect was negligible unless long incubation times were used. The absence of proteolytic activity of the molds at 4°C and the impracticality of aging beef at 18 or 24°C suggest that treatment of meat with molds to enhance tenderness may not be feasible.


2005 ◽  
Vol 187 (24) ◽  
pp. 8247-8255 ◽  
Author(s):  
S. Gingues ◽  
C. Kooi ◽  
M. B. Visser ◽  
B. Subsin ◽  
P. A. Sokol

ABSTRACT The distribution of the metalloprotease gene zmpA was determined among strains of the Burkholderia cepacia complex (Bcc). The zmpA gene was present in B. cepacia, B. cenocepacia, B. stabilis, B. ambifaria and B. pyrrocinia but absent from B. multivorans, B. vietnamiensis, B. dolosa, and B. anthina. The presence of zmpA generally correlated with extracellular proteolytic activity with the exception of five strains, which had zmpA but had no detectable proteolytic activity when skim milk agar was used as a substrate (zmpA protease deficient). Western immunoblot experiments with anti-ZmpA antibodies suggest that the zmpA protease-deficient strains do not secrete or accumulate detectable ZmpA. Transcriptional zmpA::lacZ fusions were introduced in selected strains of the Bcc. zmpA::lacZ was expressed in all strains, but expression was generally lower in the zmpA protease-deficient strains than in the zmpA protease-proficient strains. Quantitative reverse transcriptase real-time PCR demonstrated that zmpA protease-deficient strains did express zmpA mRNA, although at various levels. ZmpA has previously been shown to be positively regulated by the CepIR quorum-sensing system. Addition of exogenous AHLs did not restore extracellular protease production to any of the zmpA protease-deficient strains; however, introduction of cepR in trans complemented protease activity in two of five strains. Extracellular proteolytic activity was restored by the presence of zmpA in trans in two of the five strains. These studies suggest that although some strains of the Bcc contain the zmpA gene, multiple factors may influence its expression.


Author(s):  
Ghanyia J. Shanyoor ◽  
Fatima R. Abdul ◽  
Nehad A. Taher ◽  
Ihsan A. Raheem

About (20) Pseudomonas rogenosa isolate were experienced for their ability of protease production by calculating the diameter of lysis area after developing on skim milk agar medium (qualitatively ) and the results exhibited that only isolate no (5), was higher isolate for protease making of (26mm) of lysis area. Then, the protein concentration also identified by Bradford method and it was found of 0.16 mg/ ml , then purification was done by using an ion- exchange chromatography with DEAE sephadex G- 100 column and the results showed the presence of 1 peak of enzyme with 50 Kd of molecular weight 2 peaks of other proteins . we tried to investigate the invitro Cytotoxic effect of purified enzyme against two human cancer lines, HeP2 (Human larynx epidermed carcinoma ) , RD ( Rabdo- Sarcoma ) , and one normal cell line Ref ( Rat embryonic fibroblast ) . The cancer and normal cells were treated with different concentrations of protease enzyme ranging from ( 0.05, 0.1, 0.2, 0.4,0.8and 0.16 mg/ml) then incubated for additional 48h at 37C0 and the results showed highest toxicity ( 80.28%) of protease enzyme on RD , moderate cytotoxicity (45.52%) on Hep andslight toxicity ( 37.12% ) on normal cell line (Ref) in a concentration (0.8mg/ml).


2019 ◽  
Vol 6 (03) ◽  
Author(s):  
PK SUNDARAM ◽  
BIKASH SARKAR ◽  
UJJWAL KUMAR ◽  
AP ANURAG ◽  
DK RAGHAV ◽  
...  

About (20) Pseudomonas rogenosa isolate were experienced for their ability of protease production by calculating the diameter of lysis area after developing on skim milk agar medium (qualitatively ) andamp; the results exhibited that only isolate no (5), was higher isolate for protease making of (26mm) of lysis area. Then, the protein concentration also identified by Bradford method andamp; it was found of 0.16 mg/ ml , then purification was done by using an ion- exchange chromatography with DEAE sephadex G- 100 column andamp; the results showed the presence of 1 peak of enzyme with 50 Kd of molecular weight 2 peaks of other proteins . we tried to investigate the invitro Cytotoxic effect of purified enzyme against two human cancer lines, HeP2 (Human larynx epidermed carcinoma ) , RD ( Rabdo- Sarcoma ) , andamp; one normal cell line Ref ( Rat embryonic fibroblast ). The cancer andamp; normal cells were treated with different concentrations of protease enzyme ranging from ( 0.05, 0.1, 0.2, 0.4,0.8andamp; 0.16 mg/ml) then incubated for additional 48h at 37C 0 andamp; the results showed highest toxicity ( 80.28%) of protease enzyme on RD , moderate cytotoxicity (45.52%) on Hep andamp;slight toxicity ( 37.12% )on normal cell line (Ref) in a concentration (0.8mg/ml).


1989 ◽  
Vol 52 (12) ◽  
pp. 852-855 ◽  
Author(s):  
SEHAM A. FARRAG ◽  
ELMER H. MARTH

Autoclaved samples of skim milk were inoculated with Listeria monocytogenes (strain Scott A, California or V7), Pseudomonas fluorescens (strain P26 or B52), or a combination of L. monocytogenes plus P. fluorescens, and incubated at 7 or 13°C for 8 weeks. McBride Listeria Agar was used to determine populations of L. monocytogenes (at 0, 7, 14, 28, 42, or 56 d), and Pseudomonas isolation agar to enumerate P. fluorescens. Growth of L. monocytogenes was somewhat enhanced after 7 d of incubation at 7 but not at 13°C in the presence of pseudomonads. However, after 14 d and until the end of the incubation period (56 d), slight inactivation of L. monocytogenes in the presence of P. fluorescens was observed. L. monocytogenes did not affect growth or survival of P. fluorescens; also, no marked changes in pH of the milk were caused either by L. monocytogenes alone or by L. monocytogenes plus P. fluorescens.


2021 ◽  
Vol 11 (2) ◽  
pp. 811
Author(s):  
Federica Ianni ◽  
Alessandra Anna Altomare ◽  
Beniamino T. Cenci-Goga ◽  
Francesca Blasi ◽  
Luca Grispoldi ◽  
...  

Among various food sources, milk proteins remain the major vector for functional peptides endowed with several biological activities. Particularly, the proteolytic activity of lactic acid bacteria during milk fermentation has been one of the most followed strategies to produce bioactive peptides. In the present study, the exploration of the activity of several starter cultures, at different fermentation times, was firstly investigated by reversed phase-high performance liquid chromatography. Among the tested strains, Lactobacillus helveticus showed a higher proteolytic activity and it was submitted to further investigations by changing the fermentation substrate (skim milk, brain heart infusion, peptone water) as well as the extraction strategy (trichloroacetic acid vs. glass beads). The chromatographic analyses and the in vitro antioxidant and antihypertensive assays highlighted considerable differences for L. helveticus hydrolysates from different substrates, while a negligible impact by the two extraction protocols emerged. Furthermore, nano-high pressure liquid chromatography coupled with a high resolution mass spectrometry analyzer allowed the preliminary discrimination of fractions from fermented skim milk, likely responsible for the found activity. The obtained results suggest the possibility of varying the fermentation parameters in order to maximize the functional effects of the bioactive peptides.


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